It is known that RNA in a living organism is chemically modified, and noncoding RNA such as rRNA and tRNA is functionalized by modification. Meanwhile, mRNA is also modified and, for example, modification such as RNA editing such as A-to-I editing in which adenosine (A) is substituted by inosine (I) in the nucleotides of mRNA and C-to-U editing in which cytosine (C) is substituted by uracil (U) is known.
Among these, RNA editing is a mechanism to convert genetic information after transcription and a phenomenon to alter the base at a specific site in the base sequence of pre-mRNA generated by transcription of a gene into another base by enzyme action, and appears in various forms such as insertions, deletions or substitutions of bases (Non-Patent Document 1). This phenomenon is generally a mechanism programmed in living organisms to change gene products depending on changes in physiological conditions and environments. In higher eukaryotes, it is believed that A-to-I editing in which the adenosine (A) base at a specific site in pre-mRNA is substituted by the inosine (I) base by the action of adenosine deaminase (ADAR) has a physiologically important meaning (Non-Patent Document 2). When A-to-I base substitution occurs in an mRNA sequence encoding a protein, since the inosine (I) is read as guanosine by ribosomes during translation, codons are changed, thereby genetic information is converted and a protein having a function different from that of the original one is generated.
As the most researched RNA precursors about A-to-I editing in coding RNA, there are mentioned serotonin 2C receptor (HTR2CR) and glutamate receptor subunit B (GRIA2) (Non-Patent Document 3).
Serotonin 2C receptor (HTR2CR) is a seven-transmembrane G protein-coupled receptor mediating neural signaling in the brain by serotonin and is believed to be deeply involved in emotion control, and is known to be subjected to RNA editing (A-to-I editing) in which 5 adenosines (A) at the A to E sites on HTR2C pre-mRNA are substituted by inosine (I) by double-stranded RNA-specific adenosine deaminase (ADAR) (Non-Patent Document 4, FIG. 1).
Even in normal healthy people, adenosine (A) bases at the A site to the E site in HTR2CR mRNA are altered into the inosine (I) base with proper frequency by A-to-I editing by the catalytic action of enzymes ADAR1 and ADAR2. Consequently, it is reported that emotion is normally controlled in normal healthy people, while the regulation is broken in patients with depression and suicides. It is believed that the defect in RNA editing in serotonin 2C receptor (HTR2CR) is involved in functional psychoses such as depression, schizophrenia and autism; however, a mechanism to control normal regulation has not yet been revealed.
As shown in FIG. 2, serotonin 2C receptor (HTR2CR), when serotonin is bound to the extracellular loop of HTR2CR, transmits impulses to coupled G protein, then causes changes in properties of nerve cells via intracellular signal transduction pathway and eventually controls cerebral functions such as memory, learning and emotion. When serotonin 2C receptor (HTR2CR) is subjected to RNA editing, the amino acid sequence in the G protein-binding region of a receptor protein is changed and the signal transduction ability of the receptor is changed. In HTR2CR, up to 24 types (mainly 8 types) of receptor proteins with different amino acid sequences and different transmitting abilities are generated from a single gene by combining sites subjected to RNA editing (shown by solid-white letters in the drawing) among the base sequence of HTR2CR and amino acids to be encoded (Non-Patent Documents 4 and 5).
In the pre-mRNA of glutamate receptor subunit B (GRIA2), desensitization kinetics of the receptor and an ion channel, Ca2++ permeability, are controlled by RNA editing (Non-Patent Document 6).
As another form of RNA modification by substitution, C-to-U editing in which cytosine (C) is substituted by uracil (U) and cytosine is converted to uridine by deamination is known (Non-Patent Document 7). It is reported that the nucleus transcriptional body encoding intestinal apolipoprotein B (ApoB) is subjected to C-to-U RNA editing to convert a CAA codon to a UAA stop codon and a shorter protein than that before editing is generated (Non-Patent Document 8).
As described above, RNA editing such as A-to-I editing and C-to-U editing is involved in an important mechanism to control genetic adaptability by generating a protein different from a protein to be generated when editing does not occur. RNA modification by substitution in a coding region plays a definitive role in biological process control, and thus the development of abnormal modification is a cause of a serious disease (Non-Patent Document 9), and, in particular, the relation of RNA modification by substitution to mental disorders such as schizophrenia, bipolar disorder and major depressive disorder attracts attention.
Along with RNA precursors of serotonin 2C receptor (HTR2C) and glutamate receptor subunit B (GRIA2), particularly, γ-amino butyric acid (GABA) receptor, a receptor deeply involved in the neuropsychiatric function of the central nervous system such as potassium channels, ion channels, and other targets of RNA editing in sequences encoding proteins of other proteins have been identified recently (Non-Patent Document 10). Although high throughput sequence data suggest that RNA modification by substitution controls protein function by converting amino acid sequences, the detailed biological function of this RNA editing has not yet been revealed.
As described above, the detailed biological function of RNA editing has not yet been revealed; however, techniques for controlling site-specific RNA substitution editing remain an attractive tool to analyze and control biological processes related to RNA editing.
Ribozymes are meanwhile known as functional molecules which specifically recognize and react with a modified specific site in an RNA sequence. Among such ribozymes, a hammerhead ribozyme (HHR) which is the smallest RNA motif having catalytic action can cleave an RNA phosphodiester bond at a specific site, and the smallest hammerhead ribozyme (HHR) which cleaves a trans type is created by modifying a natural HHR (Non-Patent Document 11), and is used for suppressing in vivo target gene expression by gene control through RNA (Non-Patent Document 12). A HHR is composed of an active region having a conserved core sequence with catalytic activity at the central portion (Helix II), and recognition regions having two hybridizing arm sequences which recognize target sequences existing on the 3′ side and 5′ side of the active region (Helix III and Helix I, respectively).
A target-specific hammerhead ribozyme (HHR) can be produced by converting the hybridizing arm sequence corresponding to target RNA according to simple Watson-Crick base pairing rule. A HHR with variously different core sequences corresponding to target RNA can be designed and can be bound to target RNA by using a sequence complementary to a hybridizing sequence including a specific triplet of target RNA, and can cleave the phosphodiester bond existing on the 3′ side of the triplet (Non-Patent Document 13).
Such hammerhead ribozyme (HHR) can in vitro and in vivo cleave mRNA in a mutation-specific manner (Non-Patent Document 14). These mutation-specific properties of HHR are produced based on the triplet to be preferentially cleaved. As described above, some HHRs which can cleave target RNA in a mutation-specific manner are created; however, a HHR which can specifically recognize RNA modification by substitution has not been reported.